CN106928275B

CN106928275B - Preparation method of spiro amine aryl phosphorus oxide compound and intermediate and crystal form thereof

Info

Publication number: CN106928275B
Application number: CN201611178180.8A
Authority: CN
Inventors: 丁照中; 陈曙辉; 刘希乐; 万海文; 张路
Original assignee: Qilu Pharmaceutical Co Ltd
Current assignee: Shandong Anhong Pharmaceutical Co ltd; Qilu Pharmaceutical Co Ltd
Priority date: 2015-12-29
Filing date: 2016-12-19
Publication date: 2020-10-02
Anticipated expiration: 2036-12-19
Also published as: CN106928275A

Abstract

The invention discloses aThe invention also discloses a preparation method and a crystal form of the high-purity spiro amine aryl phosphorus oxide compound, and a method for preparing the compound shown in the formula (I) and an intermediate compound thereof.

Description

Preparation method of spiro amine aryl phosphorus oxide compound and intermediate and crystal form thereof

Technical Field

The invention relates to a preparation method and a crystal form of a high-purity spiro amine aryl phosphorus oxide compound, and also relates to a method for preparing a compound shown in a formula (I) and an intermediate compound thereof.

Background

PCT/CN2015/082605 describes a novel class of oral ALK inhibitors for the treatment of Anaplastic Lymphoma Kinase (ALK) positive metastatic non-small cell lung cancer (NSCLC), including non-small cell lung cancer that is potent and resistant to Crizotinib. The structure is shown as formula (A):

crizotinib is the first ALK inhibitor approved by the FDA for the treatment of ALK-positive lung cancer. Although the initial response by Crizotinib is very effective, most patients relapse in the first year of treatment due to development of drug resistance. On 29 months 4 2014, FDA approved certiniib for the treatment of Anaplastic Lymphoma Kinase (ALK) positive metastatic non-small cell lung cancer (NSCLC), including patients who are effective as well as resistant to crizotinib. Still other compounds are being used in clinical studies for the treatment of cancer, such as alectinib, AP-26113, and the like. Certain heterocyclic compounds have also been disclosed for use in the treatment of various cancers.

Disclosure of Invention

The invention provides a process for the preparation of a compound of formula (I),

which comprises the following steps:

wherein the content of the first and second substances,

r is selected from C_1-5Alkyl, preferably selected from methyl, ethyl, propyl, isopropyl, butyl;

R₁selected from amino protecting groups;

x is selected from F, Cl, Br, I, OMs;

the acid is selected from organic acid or inorganic acid;

the reducing agent is selected from a heavy metal catalyst-hydrogen reduction system.

In some embodiments of the invention, R is selected from: methyl, ethyl, propyl, isopropyl, butyl.

In some embodiments of the invention, R is as defined above₁Selected from: boc, Cbz, Fmoc, Alloc, Teco, methoxycarbonyl, ethoxycarbonyl, benzyl, p-methoxybenzyl.

In some embodiments of the invention, the acid is selected from: acetic acid, trifluoroacetic acid, methanesulfonic acid, trifluoromethanesulfonic acid, p-toluenesulfonic acid, camphorsulfonic acid, hydrochloric acid or sulfuric acid.

In some embodiments of the present invention, the reducing agent is selected from: palladium carbon-hydrogen, raney nickel-hydrogen, palladium hydroxide-hydrogen.

In some embodiments of the present invention, the above preparation method comprises the following steps:

wherein the content of the first and second substances,

the base is selected from alkali metal bases, alkaline earth metal bases, organometallic bases or organic bases.

In some embodiments of the present invention, the alkali metal base is selected from the group consisting of lithium hydroxide, sodium hydroxide, potassium hydroxide, cesium hydroxide, sodium carbonate, potassium carbonate, cesium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydride, and potassium hydride.

In some embodiments of the invention, the alkaline earth metal base is selected from calcium hydride.

In some embodiments of the present invention, the organometallic base is selected from sodium methoxide, lithium tert-butoxide, sodium tert-butoxide, potassium tert-butoxide, sodium ethoxide, and/or aluminum isopropoxide.

In some embodiments of the invention, the organic base is selected from diisopropylethylamine, triethylamine, N-methylmorpholine, 1, 8-diazabicycloundec-7-ene.

in some embodiments of the present invention, the above intermediate (e) can also be prepared by the following method:

in some embodiments of the invention, the compounds of formula (I) above may also be prepared by:

wherein R is₂Is selected from H or C_1-5An alkyl group;

r is as described above.

In some embodiments of the invention, R is as defined above₂Selected from H, methyl, ethyl, propyl, butyl.

In some embodiments of the present invention, the above preparation method further comprises the following reaction scheme:

in some embodiments of the present invention, the above preparation method further comprises the following reaction

The present invention also provides compounds of the formula:

the invention also provides a crystal form A of the compound shown in the formula (II), and an XRPD pattern of the crystal form A is shown in figure 1.

In some embodiments of the invention, the XRPD pattern analysis data for form a of the compound of formula (ii) above is shown in table-1.

TABLE-1 XRPD pattern analysis data for form A of the compound of formula (II)

In some schemes of the invention, the preparation method of the crystal form A comprises the steps of adding the compound shown in the formula (II) into an organic solvent or a mixed solvent of the organic solvent and water, heating to 30-reflux temperature for dissolving, and then cooling to 0-20 ℃ within 0.5-10 hours to precipitate crystals.

In some embodiments of the present invention, the above method for preparing form a, wherein the organic solvent is selected from alcoholic solvents.

In some embodiments of the present invention, in the preparation method of the form a, the alcohol solvent is a single solvent selected from methanol, ethanol, isopropanol and/or n-butanol, or a mixed solvent of several solvents.

In some embodiments of the present invention, the mixed solvent of the organic solvent and water is preferably selected from ethanol/water and methanol/water.

In some embodiments of the invention, the volume ratio of ethanol to water is 1: 0.05-10.

Definitions and explanations

As used herein, the following terms and phrases are intended to have the following meanings unless otherwise indicated. A particular phrase or term should not be considered as ambiguous or unclear without special definition, but rather construed in a generic sense. When a trade name appears herein, it is intended to refer to its corresponding commodity or its active ingredient.

The intermediate compounds of the present invention may be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combinations thereof with other chemical synthetic methods, and equivalents thereof well known to those skilled in the art, with preferred embodiments including, but not limited to, the examples of the present invention.

The chemical reactions of the embodiments of the present invention are carried out in a suitable solvent that is compatible with the chemical changes of the present invention and the reagents and materials required therefor. In order to obtain the compounds of the present invention, it is sometimes necessary for a person skilled in the art to modify or select the synthesis steps or reaction schemes based on the existing embodiments.

An important consideration in any synthetic route planning in the art is the selection of suitable protecting groups for reactive functional groups, such as amino groups in the present invention. Greene and Wuts (protective groups In Organic Synthesis, Wiley and Sons,1991) are the authorities of this area for trained practitioners. All references cited herein are incorporated herein in their entirety.

The present invention will be specifically described below by way of examples, which are not intended to limit the present invention in any way.

All solvents used in the present invention are commercially available and can be used without further purification. The reaction is generally carried out under inert nitrogen in an anhydrous solvent. Proton NMR data were recorded on a Bruker Avance III 400(400MHz) spectrometer with chemical shifts expressed as (ppm) at the low field of tetramethylsilane. Mass spectra were measured on an agilent 1200 series plus 6110(& 1956A). LC/MS or Shimadzu MS contain a DAD: SPD-M20A (LC) and Shimadzu Micromass2020 detector. The mass spectrometer was equipped with an electrospray ion source (ESI) operating in either positive or negative mode.

The invention employs the following abbreviations: DCM represents dichloromethane; Pd/C represents palladium carbon; DMF represents N, N-dimethylformamide; EtOH stands for ethanol; MeOH represents methanol; cbz represents benzyloxycarbonyl, an amine protecting group; boc for tert-butylcarbonyl is an amine protecting group; fmoc represents fluorenyl-methoxycarbonyl, which is an amine protecting group; alloc represents an allyloxycarbonyl group, an amine protecting group; teoc represents trimethylsiloxyethyl carbonyl, which is an amine protecting group; HOAc represents acetic acid; THF represents tetrahydrofuran; boc₂O represents di-tert-butyl dicarbonate; DIPEA stands for diisopropylethylamine; DIEA stands for diisopropylethylamine; DBU represents 1, 8-diazabicycloundec-7-ene; tol represents toluene; THF represents tetrahydrofuran; i-PrOH represents 2-propanol; mp represents melting point; NMP stands for N-methylpyrrolidone; AcOH represents glacial acetic acid; con. hcl (aq) stands for concentrated aqueous hydrochloric acid.

The compound is made by hand or

The software names, and the commercial compounds are under the supplier catalog name.

The powder X-ray diffraction (XRPD) method of the invention

Light pipe: cu, K-Alpha, (u, K-

)

Voltage of light pipe: 40kV, light tube current: 40mA

Scattering slit: 0.60mm

Detector slit: 10.50mm

Backscatter slit: 7.10mm

Scanning range: 4-40deg

Step length: 0.02deg

Rate: 0.1S

Sample pan rotation speed: 15rpm

Differential thermal analysis (DSC) method of the present invention

The instrument model is as follows: TA Q2000 differential scanning calorimeter

And (3) testing conditions are as follows: a sample (-1 mg) was placed in a DSC aluminum pan for testing by the method: the temperature is 25-350 ℃, and the heating rate is 10 ℃/min. The present invention is a Thermal Gravimetric Analysis (TGA) method

The instrument model is as follows: TA Q5000IR thermogravimetric analyzer

And (3) testing conditions are as follows: a sample (2-5 mg) is placed in a TGA platinum pan for testing, and the method comprises the following steps: the room temperature is 350 ℃ below zero, and the heating rate is 10 ℃/min.

The technical effects are as follows:

the process for synthesizing the compound of the formula (I) and the intermediate thereof has the following beneficial effects: the method has the advantages of low price of the initial raw materials, excellent route, low toxicity and danger of the used reagents, mild reaction conditions, simple separation and purification, easy operation and easy industrial production.

Specifically, the method comprises the following steps:

1) the raw materials of the method for preparing the compound shown in the formula (I) are conventional or common reagents, and are easily available in the market and low in price;

2) the intermediate compound (e) can be converted into two functional groups by a new intermediate compound (d) through hydrogenation reaction in one step, so that the reaction efficiency is effectively improved, and the yield is high;

3) the reaction intermediates in each step are easy to post-treat and purify, and are suitable for industrial production;

4) the A crystal form of the compound shown in the formula (II) has stable property and good patent medicine prospect.

Therefore, the invention has high industrial application value and economic value in the aspect of preparing the compound shown in the formula (I) and the intermediate thereof.

Drawings

FIG. 1 is an XRPD spectrum of Cu-Ka radiation of crystal form A of the compound of formula (II).

FIG. 2 is a DSC of form A of the compound of formula (II).

Figure 3 is a TGA profile of form a of the compound of formula (ii).

Detailed Description

For better understanding of the present invention, the following description is given with reference to specific examples, but the present invention is not limited to the specific embodiments.

Scheme 1

Example 1

Dimethylphosphineoxide

N₂A solution of diethyl phosphite (400g, 2.9mol, 1 eq) in THF (4L) was cooled to 0 deg.C under protection, and a solution of MeMgBr in ether (3M,2.9L, 3eq) was added dropwise while maintaining the temperature of the reaction solution at no higher than 20 deg.C. After the addition was complete, the mixture was allowed to warm to 20 ℃ and stirred for 0.5h, and TLC (DCM: MeOH ═ 20:1) showed completion of the reaction). The reaction solution was slowly added to an aqueous potassium carbonate solution (1184g/1448mL) to quench, with an exotherm occurring during quenching, and the temperature was controlled to be 40 ℃ or lower. A white solid precipitated, the mixture was filtered, the filter cake was washed with ethanol, the filtrate was concentrated, and the solid that appeared during the concentration was filtered. To obtainThe desired product (144.09g, 63.66% yield) was a colorless viscous oil.¹HNMR(400MHz,CDCl₃)：，7.74-7.67(m,0.5H),6.57-6.52(m,0.5H),1.56(d,J＝3.6Hz,3H),1.53(d,J＝3.6Hz,3H).

Example 2

(2-aminophenyl) dimethylphosphine oxide

2-iodoaniline (990.01g, 4.52mol, 1 eq.) and dimethylphosphine oxide (690g,5.2mol, 1.15 eq.), K₃PO₄(1.15Kg,5.42mol, 1.2 equiv.), Xantphos (130.77g,0.226mol, 0.05 equiv.) and palladium acetate (50.74g,0.226mol, 0.05 equiv.) were placed in DMF (5.5L) and water (0.55L), and the reaction mixture was heated to 110 ℃ and 125 ℃ under nitrogen and stirred for 3 hours. LCMS and TLC (PE: EA ═ 10:1, DCM: MeOH ═ 10:1) showed reaction completion. The mixture was filtered and concentrated to remove DMF, and the resulting residue was diluted with ethyl acetate (2L). Then HC/EA (4M, 2L) was added to salify the product, a large amount of solid precipitated out of the system, stirred to disperse it evenly, the solid was filtered, the filter cake was washed with acetone (0.4L x3), and the solid was dried to give the title compound (817g, yield 79.96%) as a pale yellow solid.¹HNMR(400MHz,CDCl₃):7.20(t,J＝7.6Hz,1H),7.04(dd,J＝13.6,7.6Hz,1H),6.69-6.58(m,2H),5.35(br s,2H),1.75(s,3H),1.71(s,3H).LCMS(ESI)(10-80CD):m/z:170.1[M+1].

Example 3

(2- ((2, 5-dichloropyrimidin-4-yl) amino) phenyl) dimethylphosphine oxide

To a solution of (2-aminophenyl) dimethylphosphine oxide hydrochloride (900g, 4.16mol) in ethanol (600mL) was added DIPEA (1.24Kg, 1.67L, 9.56mol, 2.3 equivalents), and the solution was observed to become clear, followed by the addition of 2,4, 5-trichloropyrimidine (915.23g, 4.99mol, 1.2 equivalents). The reaction mixture was heated to 80 ℃ and stirred for 18 hours. Tlc (etoac) showed the reaction was complete. The reaction mixture was concentrated while the reaction was cooled, and water (2L) and DCM (2L) were added and stirred for 0.5 h. After separation, the aqueous phase was extracted with DCM (500mLx3), and the combined organic phases were washed with saturated brine (2L), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. Concentrating to obtain brown solid, and pulping and washing the crude product with methyl tert-butyl etherWashing and filtration gave the title compound as a white solid (1.04Kg, yield 79%).¹H NMR(400MHz,CD₃OD):8.50(dd,J＝8.0,4.0Hz,1H),8.35-8.28(m,1H),7.69-7.59(m,2H),7.36-7.28(m,1H),1.91(s,3H),1.88(s,3H).LCMS(ESI)(5-95AB):m/z:315.9[M+1].

Scheme 2

Example 4

9-benzyl-2, 4-dioxo-3, 9-diazaspiro [5.5] undecane-1, 5-dinitrile

Ammonium acetate (122.2g,1.59mol,0.10 eq) was added to a solution of cyanoacetate (5.38kg, 47.56mol, 3eq) in methanol at 5-8 ℃; 1-benzylpiperidine 4-one (3.0kg,15.85mol,1 eq.) was then added to the reaction mixture; then, aqueous ammonia (3.42L, 25%, 22.19mol,1.4 eq.) was added to the reaction mixture at below 10 ℃ and the mixture was stirred at 0-5 ℃ for 1 hour. The reaction mixture was then warmed to 25 ℃ (room temperature) and stirred for 20 hours. White solid is separated out in the reaction system. The reaction was heated to 50 ℃ and the pH was adjusted to 4-5 with concentrated HCl (12M) while maintaining the temperature at 70 ℃ or below. The suspension was not easily stirred, ethanol and water were added, the reaction solution was cooled to 10 ℃ with stirring, stirred for 30 minutes and filtered. The filter cake was washed with water and dried to give the title compound (4Kg, 77.73% yield) as a white solid. LCMS (ESI) (0-30AB) M/z 323.0[ M +1].

Example 5

1 '-benzyl-3, 7-diazaspiro [ bicyclo [3.3.1] nonane-9, 4' -piperidine ] -2,4,6, 8-tetraone

To an 88% sulfuric acid solution (6L, prepared by adding 5.25L of concentrated sulfuric acid to 750mL of water, with vigorous exotherm on dilution) was added 9-benzyl-2, 4-dioxo-3, 9-diazaspiro [5.5] with stirring]Undecane-1, 5-dinitrile (3.0kg,9.31mol,1 eq.) fed at a temperature of less than 60 ℃ and after the addition the reaction mixture is stirred at 60-90 ℃ for 2 hours. Then water (1.2L) was added to the reaction solution, a vigorous exotherm was generated, the reaction solution was heated to 90-100 ℃ and stirred for 1.5 hours, a large amount of solids precipitated during stirring. Then, the product is processedWater (15L) was added to the reaction mixture, and the reaction solution was cooled to 30 ℃, stirred at 30 ℃ for 12 hours, and then filtered. The filter cake was washed with cold water, the solid collected and dried to give the title compound as a white solid (3.1kg, 90% purity, 88% yield).¹H NMR(400MHz,CDCl₃):11.87(s,2H),9.56(br.s.,1H),7.47(s,5H),4.34(d,J＝4.4Hz,2H),3.75(br.s.,1H),3.22(br.s.,4H),1.88(br.s.,4H).

Example 6

9-benzyl-3, 9-diazaspiro [5.5] undecane-2, 4-dione

To an aqueous NaOH solution (5M, 22.5L, 8 equivalents) at 15 deg.C was added 1' -benzyl-3, 7-diazaspiro [ bicyclo [3.3.1]]Nonane-9, 4' -piperidines]2,4,6, 8-tetraone (5.4kg, 14.08mol, 1 eq), then the mixture was warmed to 80 ℃ and stirred for 2 hours. The conversion of the amide of one ring of the starting material to the carboxylic acid was checked, the mixture was cooled to 60 ℃ and then concentrated hydrochloric acid was added slowly until the pH of the solution was around 7. The mixture was heated to 70-75 ℃ and concentrated HCl was added dropwise to control the rate of carbon dioxide release until the pH was adjusted to about 3-4. The mixture was heated to 75-85 ℃ and reacted further for 1 hour. After cooling the resulting suspension to 25 ℃ and stirring for 16 hours, the suspension was cooled to 10 ℃, the solid was filtered and washed with cold water. Both the product and the non-decarboxylated product are partially soluble in water, and the non-decarboxylated by-product can be removed by washing with water. The solid was dried to give the title compound as a white solid (2.67kg, purity 95%, yield 58%).¹H NMR(400MHz,CDCl₃):10.89(s,1H),10.60(br.s,1H),7.57(br.s.,2H),7.44(br.s.,3H),4.28(br.s.,2H),3.11(br.s.,4H),2.76(br.s.,2H),2.42(br.s.,2H),2.00-1.48(m,4H).

Example 7

3-benzyl-3, 9-diazaspiro [5.5] undecane

Slowly to 9-benzyl-3, 9-diazaspiro [5.5] at 60-70 deg.C (too slow reaction at low temperature followed by risk of temperature rise)]Lithium aluminum hydride (750g, 19.8mol, tablet, 4 equiv.) was added to a solution of undecane-2, 4-dione (1.5kg,4.86mol,1 equiv.) in tetrahydrofuran (12L), and the mixture was stirred at 70 ℃ for 16 hours, to keep the reaction solutionA few small particles of lithium aluminium hydride are seen. The mixture was cooled to 10 ℃ and quenched by the addition of water (750mL), which precipitated a large amount of solid and was difficult to stir, and ethyl acetate (5L) was added to stir the suspension followed by aqueous sodium hydroxide (1M, 750 mL). After 2 hours of continued stirring after quenching, the mixture was filtered and the filtrate was concentrated to give the title compound (750g, 50% yield) as a pale yellow oil which solidified upon cooling and was taken on to the next step without further purification.¹H NMR(400MHz,CDCl₃):7.34-7.27(m,5H),3.52(s,2H),2.88-2.73(m,4H),2.46-2.35(m,4H),1.58-1.51(m,4H),1.48-1.39(m,4H).

Scheme 3

Example 8

3-benzyl-9- (3-methoxy-4-nitrophenyl) -3, 9-diazaspiro [5.5] undecane

To 5-fluoro-2-nitrobenzyl ether (1.0kg, 5.84mol, 1 eq.) and 3-benzyl-3, 9-diazaspiro [5.5]]N, N-diisopropylethylamine (1.13kg, 8.77mol, 1.55 equiv.) was added to a solution of undecane (1.70kg, 5.57mol, 0.95 equiv.) in N-methylpyrrolidone (4L) and the reaction was stirred at 100 ℃ for 4 hours. TLC (dichloromethane: methanol ═ 6:1, Rf ═ 0.5) showed complete reaction. Cooling the reaction solution to room temperature, slowly adding 16L of water into the reaction solution to precipitate a large amount of solid, continuously stirring the suspension for 1 hour, filtering, adding the filter cake into 5L of ethanol, refluxing and pulping for 1 hour, cooling to room temperature, and filtering. The filter cake was again slurried with ethanol (5L) under reflux, then filtered and the filter cake dried to give the product (1.92kg, 83% yield) as a yellow solid.¹H NMR(400MHz,CDCl₃):8.03(d,J＝9.2Hz,1H),7.37-7.28(m,5H),6.42(dd,J＝9.6,2.8Hz,1H),6.30(d,J＝2.4Hz,1H),3.96(s,3H),3.55(s,2H),3.45-3.34(m,4H),2.53-2.38(m,4H),1.66-1.58(m,8H).

Example 9

3-benzyl-9- (3-methoxy-4-nitrophenyl) -3-methyl-3, 9-diazaspiro [5.5] undec-3-ium

2.5L of the mixture isMethyl chloride was added to a 5L kettle (20 ℃), 0.5kg of compound (c) was added to the reaction flask (20 ℃), the temperature was programmed to 25 ± 5 ℃, and stirring was carried out until the mixture was dissolved and clarified (T ═ 25 ± 5 ℃). 0.27Kg (1.5 equivalents) of iodomethane was added dropwise to the reaction mixture over 20 + -5 min (T25 + -5 deg.C), and after the addition was complete, the mixture was stirred at 25 + -5 deg.C for 6-8 hours, and a yellow solid was continuously precipitated during the reaction. Sampling and check (TLC) while stirring (TLC: 0.5ml of the reaction solution was added to 2.5ml MeOH/2.5ml DCM, developing agent DCM: MeOH: 10:1, and the reaction was stopped by monitoring the disappearance of the starting material under UV). After completion of the reaction, the reaction solution was filtered under reduced pressure. The filter cake was slurried with 1.327Kg of dichloromethane at 25. + -. 5 ℃ for 2 hours. And drying the filter cake at 50-60 ℃ in vacuum to constant weight.¹H NMR(400MHz,DMSO-d6):7.90(d,J＝9.6Hz,1H),7.65-7.46(m,5H),6.64(dd,J＝10.8,2.8Hz,1H),6.52(d,J＝2.0Hz,1H),4.65(s,2H),3.92(s,3H),3.57-3.39(m,6H),3.31-3.20(m.,2H),2.96(s,3H),2.01-1.54(m,8H).

Example 10

2-methoxy-4- (9-methyl-3, 9-diazaspiro [5.5] undec-3-yl) aniline

1.028kg (1L) of NMP was charged to a 2L autoclave (RT), 0.1kg of compound (d) was charged to the Reactor (RT), 33.52g (3eq) of HOAc was charged to the Reactor (RT), and N2 was bubbled-5 min. 10g of wet Pd-C (50%) was added to the reaction solution, and the reaction solution was purged 4 times with H2 gas. Adjusting the reaction pressure to 1.5MPa, and heating the reaction to 80-90 ℃ under the stirring condition. Stirring at 80-90 deg.C for 10 hr (when R1 pressure is less than 1.0MPa, adding H₂To 1.5MPa), central control sampling test (HPLC), sampling test (sampling method: 0.1ml was taken and dissolved in 1ml MeOH). Stopping the reaction, reducing the reaction temperature to room temperature, and then reducing the pressure of the high-pressure kettle to normal pressure; the reaction solution was filtered through celite (50g) under reduced pressure; the filter cake was washed 1 time with 0.3L DCM and the filtrate was adjusted to pH 2 with aq.hcl (1M, 0.4L); DCM (1.32Kg × 5) was added to the filtrate for extraction and TLC detection (sampling: 0.1ml of aqueous phase taken directly; developing agent: DCM/MeOH ═ 10/1, iodine development, essentially no NMP was observed). NaOH (5M, 167g) was added to the aqueous phase to adjust the pH to 10, a solid precipitated and stirring was continued for 0.5 h. Suction filtration was carried out under reduced pressure, the filter cake was washed with water (0.01L), and the filter cake was collected. Vacuum drying the solid at 60 deg.C for 5h, and checking the intermediateMeasurement (HPLC), as HPLC>At 90%, it can be directly used in the next step.¹HNMR(400MHz,CD₃OD):6.72(d,J＝8.4Hz,1H)6.64(d,J＝2.0Hz,1H)6.51(dd,J＝8.4,2.0Hz,1H)3.86(s,3H)2.95-3.08(m,4H)2.37-2.59(m,4H)2.30(s,3H)1.50-1.77(m,8H)

Example 11

(2- ((5-chloro-2- ((2-methoxy-4- (9-methyl-3, 9-diazaspiro [5.5] undec-3-yl) phenyl) amino) pyrimidin-4-yl) amino) phenyl) dimethyloxy) dimethyl

NMP (102.6g) was charged into a reaction flask (room temperature), 20g of compound (e) was charged into the reaction flask with stirring, 21.85g of compound (f) was charged into the reaction flask, and 19.93g (3eq) of MeSO was added₃H drop into reaction flask (temperature control)<40℃)N₂Bubbling for 15-20 min. Heating the reaction to 85-90 deg.C, reacting at 85-90 deg.C for 12 hr, performing central control sampling (HPLC), and sampling (detection method: dissolving 0.1ml of reaction solution in 2ml of MeOH) to obtain compound (f)<At 2.5%, the reaction was stopped. To the reaction solution was added NaOH solution (1M, 287g) to adjust pH to 13, and a large amount of solid was precipitated and stirred for 2 h. The solid was filtered and the filter cake was washed with water (40g) until the filtrate was colourless. Collecting a filter cake, filtering, solidifying and drying (55-60 ℃) to obtain an off-white crude product, adding methanol (268g) into a reaction bottle, then adding the obtained solid, heating to 60 ℃ to dissolve, adding active carbon (5.2g) into the reaction bottle, then stirring for 2.5h at 60 ℃, cooling to 30 ℃, filtering, and concentrating the mother liquor under reduced pressure to obtain an off-green solid. MeOH (155.5g) was added to the reaction flask, followed by the solid obtained above, and heated to reflux until clear. Adding 388g of purified water into the reaction bottle, naturally cooling to 15-20 ℃, and separating out white solids. Filtration and filtration were carried out, and the solid was washed once with purified water (194 g). The filter cake was collected and dried to give the compound of formula (II) (32.5 g).¹H NMR(400MHz,CD3OD):8.36(dd,J＝8.0,4.4Hz,1H),8.03(s,1H),7.69(d,J＝8.8Hz,1H),7.65-7.55(m,1H),7.51(dd,J＝8.0 8.0Hz,1H),7.32-7.20(m,1H),6.66(d,J＝2.4Hz,1H),6.45(dd,J＝8.8,2.4Hz,1H),3.85(s,3H),3.18-3.06(m,4H),2.54-2.38(m,4H),2.30(s,3H),1.85(d,J＝13.6Hz,6H),1.74-1.54(m,8H).

Scheme 4

Example 12

Tert-butyl ester 9- (3-methoxy-4-nitrophenyl) -3, 9-diazaspiro [5.5] undecane-3-carboxylic acid methyl ester

To tert-butyl-3, 9-diazaspiro [5.5]]To a solution of undecane-3-carboxylic acid ester (5.00g,1 eq) and 4-fluoro-2-methoxy-1-nitrobenzene (4.04g,1.2 eq) in acetonitrile (50.00mL) was added potassium carbonate (4.08g,29.49mmol,1.5 eq), and the reaction was stirred at 85 ℃ for 5 hours TLC (DCM: MeOH ═ 10:1, Rf of spiro cyclic amine ═ 0.5) showed completion of the reaction, and to the reaction was added water (30mL) and concentrated. After the organic solvent is evaporated, a solid is separated out. The mixture was filtered and the filter cake was purified by petroleum ether: beating the ethyl acetate mixed solvent (volume ratio is 5:1, 24mL), filtering, and adding petroleum ether: the ethyl acetate mixed solvent (volume ratio 20:1, 21mL) was slurried, filtered, and the filter cake was dried to give the product (7.5g, 94% yield) as a yellow solid.¹H NMR(400MHz,CD3OD):,7.95(d,J＝9.2Hz,1H),6.55(dd,J＝9.6,2.4Hz,1H),6.48(d,J＝2.4Hz,1H),3.95(s,3H),3.56-3.41(m,8H),1.72-1.65(m,4H),1.58-1.50(m,4H),1.48(s,9H).

Example 13

Tert-butyl-9- (4-amino-3-methoxyphenyl) -3, 9-diazaspiro [5.5] undecane-3-carboxylic acid ester

To tert-butyl-9- (3-methoxy-4-nitro-phenyl) -3, 9-diazaspiro [5.5] at room temperature]To a solution of undecane-3-carboxylic acid ester (4.00g,1 equiv.) and ammonium chloride (5.27g,10 equiv.) in methanol (50mL) was added zinc powder (6.45g,10 equiv.). Then stirred at room temperature for 15 minutes. TLC (petroleum ether: ethyl acetate 1:1, starting material Rf 0.6, product Rf 0.4) showed completion of the reaction. The reaction solution was filtered, the filtrate was concentrated, water (30mL) and saturated sodium carbonate solution (100mL) were added, the mixture was extracted three times with dichloromethane (50 mL/time), the combined organic layers were dried over anhydrous sodium sulfate, and the mixture was concentrated to give the title compound as a dark purple oil (3.12g, yield 84%).¹HNMR(400MHz,CDCl₃):6.66(d,J＝8.8Hz,1H),6.55(d,J＝2.4Hz,1H),6.45(dd,J＝8.8,2.4Hz,1H),3.85(s,3H),3.49-3.35(m,4H),3.10-2.94(m,4H),1.74-1.63(m,4H),1.56-1.42(m,13H).

Example 14

(2- ((5-chloro-2- ((2-methoxy-4- (3, 9-diazaspiro [5.5] undecan-3-yl) phenyl) amino) pyrimidin-4-yl) amino) phenyl) dimethyl phosphine oxide

To a solution of tert-butyl-9- (4-amino-3-methoxyphenyl) -3, 9-diazaspiro [5.5] undecane-3-carboxylate (80mg,0.213mmol) in tert-butanol (3.0mL) was added (2- ((2, 5-dichloropyrimidin-4-yl) amino) phenyl) dimethylphosphine oxide (67mg,0.213mmol) and methanesulfonic acid (119mg,1.07mmol) at room temperature. Then, the reaction solution was heated to 90 ℃ under nitrogen protection and stirred for 16 hours. The reaction was concentrated and isolated by preparative isolation to give the title compound as a yellow solid (25mg, 21% yield). LCMS (ESI) (5-95AB) M/z 555.2[ M +1].

Example 15

(2- ((5-chloro-2- ((2-methoxy-4- (9-methyl-3, 9-diazaspiro [5.5] undecan-3-yl) phenyl) amino) pyrimidin-4-yl) amino) phenyl) dimethylphosphineoxide

To (2- ((5-chloro-2- ((2-methoxy-4- (3, 9-diazaspiro [5.5 ]) at room temperature]Undecane-3-yl) phenyl) amino) pyrimidin-4-yl) amino) phenyl) dimethylphosphine oxide (10mg,0.182mmol) in methanol (5.0mL) was added 37% aqueous formaldehyde (30mg,0.36mmol) followed by sodium cyanoborohydride (22mg,0.36mmol), stirred at room temperature for 1 hour and LCMS detected completion, and the reaction was quenched with (1N,0.5mL) and isolated by preparative isolation to give the title compound (4mg, 40% yield).¹H NMR(400MHz,CD₃OD):,8.28(s,1H),8.12(br.s.,1H),7.81-7.68(m,3H),7.65(d,J＝2.0Hz,1H),7.56-7.49(m,1H),7.37(d,J＝8.8Hz,1H),4.02(s,3H),3.74(br.s.,4H),3.47(d,J＝12.8Hz,2H),3.24(t,J＝12.8Hz,2H),2.93(s,3H),2.42-1.97(m,5H),1.93-1.75(m,9H).LCMS(ESI)(0-60AB):m/z:569.2[M+1].

Example 16

(2- ((5-chloro-2- ((4- (9-isopropyl-3, 9-diazaspiro [5.5] undecan-3-yl) -2-methoxyphenyl) amino) pyrimidin-4-yl) amino) phenyl) dimethylphosphineoxide

Example 14 preparation of a mixture of the isolated title compound (100mg, 0.18 mmol), sodium acetate borohydride (114 mg, 0.54 mmol), acetic acid (21.6 mg,0.36mmol) and acetone (20.9 mg,0.36mmol) in tetrahydrofuran (5.0mL) was stirred at 18 ℃ for 16 h. LCMS showed reaction complete. The solvent was removed by concentration under reduced pressure to give a crude product, which was purified by preparative HPLC to give the title compound (50mg, 47% yield) as a brown solid. LCMS (ESI) (10-80AB) M/z 597.3[ M +1].

Scheme 5

Example 17

Tert-butyl-9-methyl-3, 9-diazaspiro [5.5] undecane-3-carboxylic acid ester

To a solution of tert-butyl-3, 9-diazaspiro [5.5] undecane (120mg,1 eq) in methanol (5mL) at 0 ℃ was added 37% aqueous formaldehyde (5 eq) and the mixture was warmed to 16 ℃ and stirred for 0.5 h. Sodium borohydride acetate (89mg, 3 equiv.) was then added and stirred at 16 ℃ for 12 hours. TLC detection of all conversion of the starting material, reduced pressure concentration at 35 ℃. Dichloromethane (50mL) was added for dilution, washed 3 times with water, 2 times with saturated brine (30mL), dried over sodium sulfate, filtered, and the organic phase was concentrated to give the title compound as a colorless oil (100mg, 79% yield).

Example 18

3-methyl-3, 9-diazaspiro [5.5] undecane

To a solution of tert-butyl-9-3, 9-diazaspiro [5.5] undecane (100mg,1 eq) in dichloromethane (2mL) at 0 deg.C was added trifluoromethanesulfonic acid (2mL), followed by stirring for 45 min at 16 deg.C. TLC detection of all conversion of the starting material, reduced pressure concentration at 35 ℃. Saturated sodium carbonate solution (40mL) was added, extracted 3 times with dichloromethane (40mL), washed 2 times with saturated brine (20mL), dried over sodium sulfate, filtered and the organic phase concentrated to give the title compound as a brown oil (70mg, crude).

Example 19

3- (3-methoxy-4-nitrophenyl) -9-methyl-3, 9-diazaspiro [5.5] undecane

To 3-methyl-3, 9-diazaspiro [5.5]]To a solution of undecane (4.83g,1 eq) and 4-fluoro-2-methoxy-1-nitrobenzene (5.6g,1.55 eq) in acetonitrile (100mL) was added potassium carbonate (30g, 10.2)6 equivalents), then the reaction solution is raised to 80 ℃ and stirred for 16 hours, the reaction solution is filtered after the completion of the TLC detection reaction, and the filtrate is concentrated and purified by column chromatography (eluent is petroleum ether: ethyl acetate 10:1 to 1:1) to give the title compound as a yellow solid (9.0g, crude).¹H NMR(400MHz,CD₃OD):7.95(d,J＝9.6Hz,1H),6.56(dd,J＝9.2Hz,J＝2.4Hz,1H),6.49(d,J＝2.4Hz,1H),3.95(s,3H),3.52–3.48(m,4H),3.09–2.91(m,4H),2.68(s,3H),1.78-1.72(m,4H),1.71-1.69(m,4H).LCMS(ESI)(0-60AB):m/z:320.2[M+1].

Example 20

2-methoxy-4- (9-methyl-3, 9-diazaspiro [5.5] undecan-3-yl) aniline

To 3- (3-methoxy-4-nitrophenyl) -9-methyl-3, 9-diazaspiro [5.5] at 10-30 ℃]Zinc powder (18.43g,281.80mmol,10.0 equiv.) was added portionwise to a solution of undecane (9.00g,1.00 equiv.) and ammonium chloride (15.07g,10.0 equiv.) in methanol (100 mL). After the addition was completed, the reaction was completed by TLC after 30 minutes of reaction at 30 ℃. The reaction solution was filtered, the filtrate was concentrated, and then a saturated sodium carbonate solution (100mL) and water (50mL) were added to conduct extraction three times with methylene chloride (80 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated to give the title compound as a dark purple oil (7.00g, 86% yield).¹H NMR(400MHz,CD₃OD):,6.72(d,J＝8.4Hz,1H),6.64(d,J＝2.0Hz,1H),6.51(d,J＝8.4Hz,1H),3.86(s,3H),3.08-2.96(m,4H),2.58-2.45(m,4H),2.32(s,3H),1.75-1.52(m,8H).

Scheme 6

Example 21

3- (3-methoxy-4-nitrophenyl) -3, 9-diazaspiro [5.5] undecane

Tert-butyl-9- (3-methoxy-4-nitrophenyl) -3, 9-diazaspiro [5.5]A mixed solution of undecane-3-carboxylic acid (0.7g, 1.73mmol) in trifluoroacetic acid (4mL) and dichloromethane (4mL) was stirred at 16 ℃ for 1 hour. LCMS showed completion of the reaction, aqueous sodium carbonate (50mL) was added to the mixture and extracted with dichloromethane (50mL × 2). the organic layer was extracted with anhydrous sulfuric acidSodium was dried, filtered and concentrated to give the title compound (0.7g, crude) as a yellow solid.¹H NMR(400MHz,CD₃OD):,7.95(d,J＝9.2Hz,1H),6.55(d,J＝8.8Hz,1H),6.48(s 1H),3.95(s,3H),3.51–3.49(m,4H),2.90–2.87(m,4H),1.70-1.66(m,4H),1.61-1.57(m,4H).LCMS(ESI)(0-60AB):m/z:306.0[M+1].

Example 22

3- (3-methoxy-4-nitrophenyl) -9-methyl-3, 9-diazaspiro [5.5] undecane

To a solution of example 21(0.7g, 2.3mmol) in tetrahydrofuran (10mL) at 16 deg.C was added aqueous formaldehyde (207mg, 6.9mmol, 37%) and stirred at 16 deg.C for 0.5 h. Sodium borohydride acetate (1.5g, 6.9mmol) was then added and stirred at 16 ℃ for 12 hours. LCMS showed reaction completion. The mixture was diluted with dichloromethane (60mL), filtered and concentrated to give the title compound (0.6g, 82% yield) as a yellow oil.¹H NMR(400MHz,CD₃OD):,7.95(d,J＝9.6Hz,1H),6.56(dd,J＝9.2Hz,J＝2.4Hz,1H),6.49(d,J＝2.4Hz,1H),3.95(s,3H),3.52–3.48(m,4H),3.09–2.91(m,4H),2.68(s,3H),1.78-1.72(m,4H),1.71-1.69(m,4H).LCMS(ESI)(0-60AB):m/z:320.2[M+1].

Example 23

2-methoxy-4- (9-methyl-3, 9-diazaspiro [5.5] undecan-3-yl) aniline

To example 22(0.6g, 1.9mmol) in EtOH/H₂To a mixture of O (12mL) were added iron powder (1.1g, 18.8mmol) and ammonium chloride (1.1g, 18.8 mmol). The mixture was stirred at 80 ℃ for 2 hours. TLC (DCM: MeOH ═ 6:1) showed the reaction was complete. The reaction mixture was filtered and concentrated. The resulting residue was purified by preparative TLC (DCM: MeOH ═ 6:1) to give the title compound (400mg, 74% yield) as a green solid. 1H NMR (400MHz, CD3OD), 6.72(d, J ═ 8.4Hz,1H),6.64(d, J ═ 2.0Hz,1H),6.51(d, J ═ 8.4Hz,1H),3.86(s,3H),3.08-2.96(m,4H),2.58-2.45(m,4H),2.32(s,3H),1.75-1.52(m,8H).

Example 24

Preparation of crystal form A of compound of formula (II)

Crude (860g) of (2- ((5-chloro-2- ((2-methoxy-4- (9-methyl-3, 9-diazaspiro [5.5] undec-3-yl) phenyl) amino) pyrimidin-4-yl) amino) phenyl) dimethylphosphineoxide and purified water (8.6Kg) were added to a reaction vessel at room temperature. Dropwise adding a hydrochloric acid solution (6M, about 700mL) into the reaction kettle, and adjusting the pH value of the solution to 2-3. Dichloromethane (2.25Kg) was added to the reaction vessel, stirred for 30 minutes and allowed to stand for stratification. After the organic layer was separated, dichloromethane (2.25Kg) was added to the reaction vessel, stirred for 30 minutes, allowed to stand for delamination, and the aqueous layer was collected.

The pH of the aqueous layer was adjusted to 10-11 with an aqueous sodium hydroxide solution (1N) to precipitate a white solid. Stirring was continued for 30 minutes and then filtered. The filter cake was dried under vacuum at 60 ℃ for 22 h to give a white solid (830 g).

The solid (526g) obtained above, ethanol (1.66Kg) and purified water (1.05Kg) were charged into a reaction vessel, and mixed with stirring. The mixture was warmed to 84 ℃ and stirred at this temperature until the mixture was clear.

The system was slowly cooled to room temperature, stirred for 16 hours and filtered. The filter cake was washed once with a mixed solvent (100g) of ethanol/water (volume ratio, 2/1). The filter cake was dried under vacuum at 60 ℃ for 21 hours to give form a of the compound of formula (ii) (472g, 99.05% purity, 89% yield) as a white solid.

Solubility test of crystal form A in different solvents

About 2mg of the sample was weighed into a 1.5mL liquid phase vial, and the following solvents were added stepwise using a pipette, respectively, and dissolved by shaking manually. The test was conducted at room temperature and the dissolution was judged by the naked eye and the results are shown in Table 2.

Table 2 solubility results of form a in different solvents

Biochemical experiments

Experimental Material

Enzyme: ALK wild type, ALK C1156Y and ALK L1196M were all purchased from Carna Biosciences (Japan), and EGFR T790M/L858R was purchased from Life technology (Madison, Wis.).

HTRF kit: substrates purchased from Cis-Bio International, including Eu-labeled TK1 antibody, XL665 and biotin-labeled TK1 polypeptide.

A detection instrument: envision (perkinelmer).

Experimental methods

Test compounds were diluted in 3-fold gradient to obtain 11 doses at final concentrations from 1uM to 0.017 nM.

10ul wild type ALK enzyme reaction mixture system: 0.5nM wild type ALK,1uM biotin-TK1peptide,30uM ATP. Reaction buffer: 50mM Hepes (pH7.5),10mM MgCl2,0.01mM NaV3VO4. the reaction plate is a whitet Proxiplate 384-Plus plate (Perkinelmer), and the reaction is carried out at room temperature for 90 minutes.

10ul ALK C1156Y enzyme reaction mixture system, 0.15nM ALK C1156Y,1uM biotin-TK1peptide,30uM ATP. Reaction buffer: 50mM Hepes (pH7.5),10mM MgCl2,0.01mM NaV3VO4. the reaction plate is white Proxiplate 384-Plus plate (Perkinelmer). The reaction was carried out at room temperature for 60 minutes.

10ul ALK L1196M enzyme reaction mixture system, 0.15nM ALK L1196M,1uM biotin-TK1peptide,30uM ATP. Reaction buffer: 50mM Hepes (pH7.5),10mM MgCl2,0.01mM NaV3VO4. the reaction plate is white Proxiplate 384-Plus plate (Perkinelmer), and the reaction is carried out at room temperature for 60 minutes.

10ul EGFR T790M/L858R enzyme reaction mixture system, 0.08nM EGFR T790M/L858R,1 uMbitin-TK 1peptide, 20uM ATP. Reaction buffer: 50mM Hepes (pH7.5),10mM MgCl2,0.01mM NaV3VO4. the reaction plate is white Proxiplate 384-Plus plate (Perkinelmer). The reaction was carried out at room temperature for 60 minutes.

Detection reaction: 10ul of detection reagent was added to the reaction plate at a final concentration of 2nM for antibodies and 62.5nM for XL 665. Incubate at room temperature for 60 minutes. Envision read plate.

Data analysis

The readings were converted to inhibition (%) (Min-Ratio)/(Max-Min) × 100% by the following formula. IC50 data were measured by 4-parameter curve fitting (Model 205in XLFIT5, iDBS).

Cell experiments

Experimental Material

RPMI1640, fetal bovine serum, penicillin/streptomycin solution, all purchased from Life Technology (Madison, Wis.). Cell Titer-Glo luminescence Cell viability reagents were purchased from Promega (Madison, Wis.). Karpas299 Cell line was purchased from European Collection of Cell Cultures (ECACC). Plate reading instrument: envision (perkinelmer).

Experimental methods

384 well plates, each of 2500 Karpas-299 cells, 45ul volume. The cells were incubated overnight at 37 ℃ in a CO2 incubator. Test compounds were diluted in 3-fold gradients to obtain 10 dose concentrations from 2.5mM to 0.127uM, in duplicate wells. The middle plate was filled with 49ul of medium per well. Transfer 1ul of compound from the gradient dilution compound plate to the middle plate and mix well. Then 5ul of liquid was taken from the middle plate and transferred to the cell plate. The cells were further cultured in a CO2 incubator for 72 hours. After 72 hours, 25ul of detection reagent was added. Incubate for 10 min at room temperature and Envision read the plate.

Data analysis

The reading was converted into an inhibition ratio (%)(Max-Sample)/(Max-Min)*100％. IC50 data were measured by 4-parameter curve fitting (Model 205in XLFIT5, iDBS).

Data for ALK enzyme inhibition IC50, ALK L1196M enzyme inhibition IC50, ALK C1156Y enzyme inhibition IC50, EGFR T790M/L858R enzyme inhibition IC50, and ALK IC50 of Karpas-299 cells of the compounds of the invention are shown in the following tables. Compounds with IC50 between 1-100nM are indicated by +++.

In vivo efficacy study

The following in vivo pharmacodynamic data show that the compound of the invention shows unexpected antitumor activity and tumor volume reduction compared with the reference compound AP26113 on both a wild type LU-01-0015 lung cancer patient-derived xenograft (PDX) model (BALB/c nude mice) and a LU-01-0319Crizotinib drug-resistant model (BALB/c nude mice). For example, in the LU-01-0015 model, representative compounds of formula (II) are23 days after administration (25 mg/kg), the tumor volume was about 277mm from the very beginning³Reduced to 30-45mm³Whereas AP26133 is reduced to only 119mm³。

1. In vivo drug effect experiment is carried out on xenograft (PDX) BALB/c nude mice with source of subcutaneous implantation LU-01-0015 lung cancer patient

BALB/c nude mice, female, 6-8 weeks, weighing about 18-22 grams, were kept in a special pathogen-free environment in a single ventilated cage (5 mice per cage). All cages, bedding and water were sterilized prior to use. All animals were free to obtain a standard certified commercial laboratory diet. There were 80 mice purchased from BK laboratory animal Co, Shanghai, LTD for study. Each mouse was implanted subcutaneously in tumor tissue (20-30 cubic millimeters) in the right flank for tumor growth. The experiment was started when the mean tumor volume reached about 250-300 cubic millimeters. The test compounds were administered orally at 25mg/kg daily. Tumor volume was measured every 3 days with a two-dimensional caliper, the volume being measured in cubic millimeters and calculated by the following formula: v ═ 0.5a × b2, where a and b are the major and minor diameters of the tumor, respectively. The anti-tumor efficacy was determined by dividing the mean tumor gain volume of animals treated with the compound by the mean tumor gain volume of untreated animals.

2. Transplanting LU-01-0319 xenograft tumor under skin, and performing in vivo drug effect experiment on Crizotinib-resistant BALB/c nude mouse

Initially, a LU-01-0319 xenograft tumor model obtained from surgically excised clinical specimens and implanted in nude mice, defined as lot P0 (LU-01-0319-P0). The next batch of tumor implantations from P0 was defined as batch P1 (LU-01-0319-P1). FP3 was recovered from P2 and the next batch was defined as batch FP4 from FP3 tumor implantation. Tumor-bearing mice were treated with Crizotinib about 2-3 weeks after tumor implantation when the tumor size reached about 300mm 3. A tumor with a sustained increase was defined as the LU-01-0319 anti-tumor model. BALB-c nude mice, female, 6-8 weeks, weighing about 18-22 grams, 75 mice were used for the study, purchased from BK Laboratory Animal co. Tumor sections of LU-01-0319R FP6 (about 30 mm) were implanted subcutaneously in the right flank of each mouse³) And is used for the growth of tumors. About 2-3 weeks after tumor implantation, the tumor size reaches about 300mm³In this case, tumor-bearing mice were treated with Crizotinib (10/25/50/75 mg/kg). The dose of Crizotinib may be varied as appropriate depending on the size of the tumor. When the average tumor volume reaches about-500 mm³Experimental studies were started. Test compounds were administered orally once daily. Tumor size was measured twice weekly, in two dimensions with calipers, and volume was calculated using the following formula: v ═ 0.5a x b2, where a and b are the major and minor diameters of the tumor, respectively. The anti-tumor efficacy was determined by dividing the mean tumor gain volume of animals treated with the compound by the mean tumor gain volume of untreated animals.

Administration amount is 3 mg/Kg; administration amount 10 mg/Kg; 25mg/Kg

3. In vivo drug effect experiment is carried out on a xenograft (PDX) BALB/c nude mouse from a patient with LU-01-0319 lung cancer implanted subcutaneously

BALB/c nude mice, female, 6-8 weeks, weighing about 18-22 grams, were kept in a special pathogen-free environment in a single ventilated cage (5 mice per cage). All cages, bedding and water were sterilized prior to use. All animals were free to obtain a standard certified commercial laboratory diet. There were 80 mice purchased from BK laboratory animal Co, Shanghai, LTD for study. Each mouse was implanted subcutaneously in tumor tissue (20-30 cubic millimeters) in the right flank for tumor growth. The experiment was started when the mean tumor volume reached about 250-300 cubic millimeters. The test compounds were administered orally at 10mg/kg daily. Tumor volume was measured every 3 days with a two-dimensional caliper, the volume being measured in cubic millimeters and calculated by the following formula: v ═ 0.5a × b2, where a and b are the major and minor diameters of the tumor, respectively. The anti-tumor efficacy was determined by dividing the mean tumor gain volume of animals treated with the compound by the mean tumor gain volume of untreated animals.

The compounds of formula (II) are useful in the treatment of a variety of cancers including anaplastic large cell lymphoma, non-small cell lung cancer, diffuse large B-cell lymphoma, inflammatory myofibroblast tumors, neuroblastoma, thyroid undifferentiated carcinoma and rhabdomyosarcoma. The compounds of formula (II) may be used as monotherapy or in combination with other chemotherapeutic agents.

Claims

1. A process for the preparation of a compound of formula (I),

which comprises the following steps:

wherein the content of the first and second substances,

r is selected from C_1-5An alkyl group;

R₁selected from amino protecting groups;

x is selected from fluorine, chlorine, bromine, iodine and methyl sulfonate;

the acid is selected from organic acid or inorganic acid;

the reducing agent is selected from palladium carbon-hydrogen, Raney nickel-hydrogen and palladium hydroxide-hydrogen.

2. The process according to claim 1, wherein R is selected from methyl, ethyl, propyl, butyl;

R₁selected from: tert-butoxycarbonyl, benzyloxycarbonyl, fluorenylmethyloxycarbonyl, allyloxycarbonyl, trimethylsiloxyethoxycarbonyl, methoxycarbonyl, ethoxycarbonyl, benzyl and p-methoxybenzyl;

the acid is selected from: acetic acid, trifluoroacetic acid, methanesulfonic acid, trifluoromethanesulfonic acid, p-toluenesulfonic acid, camphorsulfonic acid, hydrochloric acid or sulfuric acid.

3. The production method according to claim 2, wherein the propyl group is an isopropyl group.

4. The production method according to any one of claims 1 to 3, comprising the steps of:

wherein the base is selected from alkali metal bases, alkaline earth metal bases, organometallic bases or organic bases;

r, R therein₁X is as defined in claim 1.

5. The preparation method according to claim 4, wherein the alkali metal base is selected from the group consisting of lithium hydroxide, sodium hydroxide, potassium hydroxide, cesium hydroxide, sodium carbonate, potassium carbonate, cesium carbonate, sodium hydrogen carbonate, potassium hydrogen carbonate, sodium hydride, potassium hydride;

the alkaline earth metal base is selected from calcium hydride;

the organic metal base is selected from sodium methoxide, lithium tert-butoxide, sodium tert-butoxide, potassium tert-butoxide, sodium ethoxide and/or aluminium isopropoxide;

the organic base is selected from diisopropylethylamine, triethylamine, N-methylmorpholine and 1, 8-diazabicycloundecen-7-ene.

6. A process for the preparation of a compound of formula (i) according to claim 1, comprising the steps of:

wherein R is₂Is selected from H or C_1-5An alkyl group;

r, X are as claimed in claim 1 or 2.

7. The method of claim 6, wherein R₂Selected from H, methyl, ethyl, propyl, butyl.

8. The production method according to claim 6 or 7, further comprising the reaction steps of:

and/or

Wherein R is₁As defined in claim 1.

9. An intermediate for the preparation of a compound of formula (i) according to claim 1, selected from:

wherein R is₁R, X is as defined in claim 1.

10. Form a of a compound of formula (ii) having an X-ray powder diffraction pattern with characteristic diffraction peaks at the following 2 Θ angles: 17.999 degrees, 21.532 degrees, 19.009 degrees, 25.381 degrees, 18.280 degrees, 20.704 degrees, 20.330 degrees, 24.924 degrees

11. Form a of a compound of formula (ii) having an X-ray powder diffraction pattern with characteristic diffraction peaks at the following 2 Θ angles: 8.752 °, 9.602 °, 10.35 °, 11.491 °, 12.383 °, 13.978 °, 14.474 °, 14.867 °, 15.992 °, 16.799 °, 17.999 °, 18.28 °, 19.009 °, 20.33 °, 20.704 °, 21.532 °, 22.185 °, 22.834 °, 23.288 °, 23.916 °, 24.924 °, 25.381 °, 25.931 °, 26.402 °, 27.21 °, 28.373 °, 29.007 °, 29.419 °, 31.829 °, 32.264 °, 34.214 °, 38.357 °,

12. the XRPD pattern of the crystal form A of the compound shown as the formula (II) is shown as figure 1

13. The crystalline form a according to any one of claims 10-12, characterized in that: the crystal form has a DSC pattern shown in figure 2.

14. The crystalline form a according to any one of claims 10-12, characterized in that: the crystalline form has a TGA profile as shown in figure 3.

15. The preparation method of the crystal form A according to any one of claims 10 to 14, which comprises the steps of adding the compound shown in the formula (II) into an organic solvent or a mixed solvent of the organic solvent and water, heating to 30 ℃ to reflux temperature for dissolution, and then cooling to 0-20 ℃ within 0.5-10 hours to precipitate crystals; wherein the organic solvent is selected from one single solvent or a mixed solvent of several solvents of ethanol, isopropanol and/or n-butanol; the mixed solvent of the organic solvent and water is selected from ethanol/water, and the volume ratio of the ethanol to the water is 1: 0.05-10.